Impaired mitochondrial oxidative phosphorylation (OXPHOS) capacity causes complex, degenerative disorders that affect all major organs and systems including heart, skeletal muscle and the nervous system. These diseases result from mutations in either nuclear genes or mtDNA, both of which encode essential OXPHOS components. There is also a documented decline in mitochondrial OXPHOS capacity with age, which correlates with the accumulation of mtDNA mutations in normal aging tissues. Therefore, mitochondrial involvement in the aging process and in the etiology of late-onset disorders such as cardiomyopathy and the common neurodegenerative diseases, Alzheimer's and Parkinson's, is almost certain. Transcription of human mtDNA-encoded OXPHOS subunits is initiated by a single-subunit mitochondrial RNA polymerase and two transcription factors. This relatively simple transcriptional apparatus not only makes this system an excellent model in which to understand transcriptional regulation in general, but also increases the likelihood of deciphering how this process is regulated in vivo in a relatively short time frame. Such advancement will allow the contribution of mitochondrial gene expression to the overall regulation of OXPHOS to be established and perhaps facilitate development of novel ways to counteract the pathological consequences resulting from lack of proper mitochondrial gene expression in human cells. The overall goal of this project is to understand how mitochondrial RNA polymerase. transcription factors and other key regulatory proteins function to control mitochondrial gene expression. Specific Aim I is to characterize the activation properties of a recently discovered human mitochondrial transcription factor (hmtTFB) at mtDNA promoters and the relevance of its novel relationship to enzymes that methylate RNA. Specific Aim II is to elucidate subsequent steps of mitochondrial gene expression, which involve the coupling of transcription to membrane-associated events involved in translation. In both aims, we take advantage of a yeast model system and utilize the information gained to guide our studies on the corresponding human components. Altogether, it is anticipated that completion of these aims will provide important new information regarding the control of mitochondrial gene expression, which is critical in order to unravel the complex involvement of OXPHOS dysfunction in human diseases and aging.